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Biomedical subjects

M Orlowski

Publications and source records attributed to M Orlowski.

At least 91 records · Page 5Linked to original sources
PubMed1984

Identification of a thermolysin-like metalloendopeptidase in serum: activity in normal subjects and in patients with sarcoidosis.

A thermolysin-like metalloendopeptidase, optimally active at a neutral pH, was identified in human serum. The enzyme cleaves the synthetic substrate glutaryl-Ala-Ala-Phe-2-naphthylamide at the Ala-Phe bond. Activity was determined by measuring the rate of formation of Phe-2-naphthylamide in a coupled enzyme assay in the presence of excess aminopeptidase M. 2-Naphthylamine released during the reaction was determined by a diazotization procedure. Enzyme activity is not affected by inhibitors of serine, thiol, or carboxyl proteases, but is sensitive to inhibition by metal chelators such as EDTA and o-phenanthroline. Dialysis against EDTA leads to loss of activity, which can be fully restored by zinc and cobalt ions. The serum enzyme closely resembles a membrane-bound metalloendopeptidase (EC 3.4.24.11) abundant in lung, spleen, and kidney in that both enzymes are inhibited by the same active-site-directed inhibitors. In addition, an antiserum obtained against the metalloendopeptidase from rabbit kidney shows strong cross-reactivity with the serum enzyme. Metalloendopeptidase activity was measured in 150 controls and in 95 patients with sarcoidosis; the two groups had significantly different enzyme activities (p less than 0.001). The mean enzyme activity in the sarcoidosis group was more than threefold higher than that of the control group. The mean enzyme activity for patients with active disease was more than double that of patients with inactive disease and more than four times that of controls (p less than 0.001). This is noteworthy because angiotensin converting enzyme, a zinc-dipeptidyl carboxypeptidase with a mechanism of action similar to that of the metalloendopeptidase, has also been reported to be increased in the serum of patients with active sarcoidosis. Enzyme activity in patients with active tuberculosis, primary pulmonary neoplasms, and idiopathic interstitial pulmonary fibrosis did not differ significantly from that of controls.

2-Naphthylamine↗
PubMed1983

A soluble metalloendopeptidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides.

A metalloendopeptidase, optimally active at a neutral pH, was purified from the soluble fraction of brain homogenates. The enzyme (molecular weight about 67000) is strongly inhibited by metal chelators such as EDTA and o-phenanthroline. An EDTA-treated enzyme can be reactivated by several divalent metal ions including Zn2+, Co2+ and Mn2+. The specificity and kinetic parameters of the enzyme were studied with a series of model synthetic substrates. The enzyme preferentially cleaves peptide bonds in which the carbonyl group is contributed by an aromatic amino acid residue in the P1 position. The lowest Km values and the highest Kcat/Km ratios were obtained with substrates having aromatic residues in the P'3 and P1 position or in the P'3 and both the P1 and P2 positions. Lower kcat/Km ratios were obtained with substrates having arginine residues in position P1, and even lower values with those substrates having a glycine or aspartyl residue in this position. Introduction of a D-amino acid residue in either position P1 or P'1 renders the substrate totally resistant to hydrolysis. The specificity studies suggest that the active site of the metalloendopeptidase can accommodate at least five amino acid residues, with two of those residues binding on the N-terminal side and three binding on the C-terminal side of the hydrolyzed bond. Several biologically active peptides are cleaved by the enzyme at sites consistent with the specificity deduced from studies with model synthetic substrates.

Animals↗
PubMed1983

Pituitary endopeptidases.

This review summarizes our knowledge of pituitary endopeptidases. Emphasis has been placed on well-characterized enzymes and their potential roles in proteolytic processes of the pituitary. Because of space limitations, degradation of biologically active peptide by crude preparations has generally not been discussed. Only a few proteolytic enzymes are at present adequately characterized, and knowledge of their physiological function in vivo is insufficient. Among the many functions of proteolytic enzymes, those that are specific for the pituitary as an endocrine gland are of primary interest. Such functions include inactivation of neuropeptides and factors that control the secretory function of the pituitary, processing of precursors destined for secretion, selective cleavage of prohormones into active fragments, and degradation of inactive fragments. While some of the enzymes described here, such as cathepsin D, could be expected to have primarily a degradative function, others could potentially be involved in hormonal metabolism, since they exhibit trypsin-like, chymotrypsin-like, and dipeptidyl carboxypeptidase-like activities, all potentially useful in hormonal conversions. Data suggestive of the presence in the pituitary of enzymes involved in removal of the 'signal sequence', and enzymes involved in hormone processing by cleavage of bonds after a pair of basic residues and in the subsequent removal of these residues by a carboxypeptidase B-like activity have been published. None of these enzymes, however, has been isolated or purified to a degree that would allow determination of its specificity, mechanisms of action, physicochemical properties, and susceptibility to specific inhibitors. Questions that remain unresolved ask whether differences in the processing pathways in various anatomical parts of the pituitary are due to the presence of proteases with different specificities, or to different disposition of these enzymes, and factors, such as conformation of the substrate and its secondary modification, for example by glycosylation or phosphorylation. Proof of a functional involvement of a protease in hormonal processing should include demonstration that inhibition of activity results in inhibition of processing in the intact cell. Specific inhibitors of processing enzymes could potentially be used to modulate pituitary function, and thus have pharmacological interest. Although there are few answers to the above problems at present, the questions are well defined, and it can be expected that the rapidly expanding research on pituitary proteases will soon provide some of the answers.

Amidohydrolases↗
PubMed1983

Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex.

Pituitary cation-sensitive neutral endopeptidase splits peptide bonds on the carboxyl side of hydrophobic amino acids (chymotrypsin-like activity), basic amino acids (trypsin-like activity), and acidic amino acids (peptidyl-glutamyl-peptide bond hydrolyzing activity). All three activities copurify, are inhibited by cations, and reside in a single high-molecular weight soluble protein complex. Treatment with sodium dodecylsulfate and 2-mercaptoethanol dissociates this complex into five low-molecular weight components. Incubation of the complex at 37 degrees C in buffers of high ionic strength produces aggregation and progressive loss of all three activities. Experiments with inhibitors and activators indicate that the three activities are catalyzed by distinct components. Benzyloxycarbonyl-glycyl-glycyl-leucinal, a peptide aldehyde transition state analog of the substrate used to measure the chymotrypsin-like activity, exclusively inhibits that activity (Ki = 2.5 x 10(-4) M), while markedly activating the trypsin-like activity. The trypsin-like activity is inhibited by leupeptin (Ki = 1.2 x 10(-6) M) and by sulfhydryl blocking agents, and activated by thiols, suggesting that this activity is due to a thiol protease. The peptidylglutamyl-peptide hydrolyzing activity is activated almost 10-fold by low concentrations of sodium dodecylsulfate, inhibited by bovine serum albumin, and suppressed at high enzyme concentrations, suggesting that this component readily interacts with other proteins, including the complex itself. The results indicate that cation-sensitive neutral endopeptidase is a multicatalytic protease complex whose distinct proteolytic activities are associated with separate components of this high-molecular weight protein.

Animals↗
PubMed1983

Inhibition of rabbit brain prolyl endopeptidase by n-benzyloxycarbonyl-prolyl-prolinal, a transition state aldehyde inhibitor.

Prolyl endopeptidase cleaves peptide bonds on the carboxyl side of proline residues within a peptide chain. The enzyme readily degrades a number of neuropeptides including substance P, neurotensin, thyrotropin-releasing hormone, and luteinizing hormone-releasing hormone. The finding that the enzyme is inhibited by benzyloxycarbonyl-prolyl-proline, with a Ki of 50 microM, prompted the synthesis of benzyloxycarbonyl-prolyl-prolinal as a potential transition state analog inhibitor. Rabbit brain prolyl endopeptidase was purified to homogeneity for these studies. The aldehyde was found to be a remarkably potent inhibitor of prolyl endopeptidase with a Ki of 14 nM. This Ki is more than 3000 times lower than that of the corresponding acid or alcohol. By analogy with other transition state inhibitors, it can be assumed that binding of the prolinal residue to the S1 subsite and the formation of a hemiacetal with the active serine of the enzyme greatly contribute to the potency of inhibition. The specificity of the inhibitor is indicated by the finding that a variety of proteases were not affected at concentrations 150 times greater than the Ki for prolyl endopeptidase. The data indicate that benzyloxycarbonyl-prolyl-prolinal is a specific and potent inhibitor of prolyl endopeptidase and that consequently it should be of value in in vivo studies on the physiological role of the enzyme.

Animals↗
PubMed1983

Cathepsin B and prolyl endopeptidase activity in rat peritoneal and alveolar macrophages. Stimulation of peritoneal macrophages by saline lavage.

Cathepsin B and prolyl endopeptidase activity was determined in purified rat pulmonary and peritoneal macrophages and cathepsin B was determined in blood monocytes. The activity of cathepsin B in pulmonary macrophages was more than two times higher than in resident peritoneal macrophages and about eight times higher than in circulating monocytes. The simple process of peritoneal lavage with PBS induced an increase in cathepsin B activity in peritoneal macrophages that by 96 and 144 hr reached those levels observed in pulmonary macrophages. Both pulmonary and peritoneal macrophages contain prolyl endopeptidase activity. Repeated peritoneal lavage induced a significant increase in the activity of the enzyme, but not of the magnitude observed for cathepsin B. The data suggest that the transformation of monocytes into macrophages is associated with a significant increase in cathepsin B activity and that this increase is much greater in pulmonary than in peritoneal macrophages.

Animals↗
PubMed1982

Determination of specificity of endopeptidases by combined high-performance liquid chromatography and amino acid analysis.

The specificity of three neutral endopeptidases toward several biologically active peptides was determined by combined high-performance liquid chromatography and amino acid analysis of the degradation products. Incubation mixtures were chromatographed on a reversed-phase column equilibrated with a mixture of acetonitrile and potassium phosphate buffer (0.05 M; pH 2.0). Reaction products were eluted with a linear gradient of acetonitrile and the absorbance of the effluent monitored at 210 nm. Fractions corresponding to discrete peaks were subjected to quantitative amino acid analysis. The peptide bond undergoing cleavage is readily assigned from the knowledge of the primary structure of the peptide and the amino acid composition of the reaction products.

Amino Acids↗
PubMed1982

Subcellular distribution of prolyl endopeptidase and cation-sensitive neutral endopeptidase in rabbit brain.

The subcellular distribution of prolyl endopeptidase, and of cation-sensitive neutral endopeptidase, two enzymes actively metabolizing many neuropeptides, was determined in homogenates of rabbit brain. The subcellular distribution of both enzymes was more similar to lactate dehydrogenase, a cytoplasmic enzyme marker, than to choline acetyltransferase, a synaptosomal marker. Only 35% of the activity of these two neutral endopeptidases was found in the crude mitochondrial fraction (P2), the bulk of the remaining activity being associated with the high-speed supernatant. Prolyl endopeptidase and cation-sensitive neutral endopeptidase thus can be regarded as mainly cytoplasmic enzymes in the rabbit brain.

Animals↗
PubMed1982

Growth-rate-dependent adjustment of ribosome function in chemostat-grown cells of the fungus Mucor racemosus.

The dimorphic fungus Mucor racemosus was grown as a yeast in a chemostat. Cellular growth rates were varied over a fourfold range under an atmosphere of N2 and over an eightfold range under CO2. Under either atmosphere, an increase in the cellular growth rate resulted in increases in (i) the cellular ribosome concentration, (ii) the percentage of ribosomes active in protein synthesis, and (iii) the rate of polypeptide chain elongation. The rate of protein synthesis in this organism can therefore be regulated by adjustment of all of these mechanisms.

Carbon Dioxide↗
PubMed1982

Stored mRNA in sporangiospores of the fungus Mucor racemosus.

When introduced into nutrient medium under air, the asexual sporangiospores of Mucor racemosus germinated with 5 to 8 h, culminating with the emergence of germ tubes. We found that sporangiospores increased 20% in dry weight during the first 60 min of germination, indicating a high degree of synthetic activity. Sucrose density gradient analysis of spore extracts revealed that the percentage of ribosomes associated with mRNA increased from 22.5% in dormant spores to 85% within 10 min after the addition of medium and remained at this level for at least 3 h. L-[14C]leucine was immediately incorporated at a rapid rate into protein of a leucine auxotroph, whereas [3H]uracil or [32P]phosphate was incorporated into RNA at a significant rate only 20 min after the addition of medium. This newly synthesized RNA occurred in polysomes only after 30 min had passed. Pool synthesized RNA occurred in polysomes only after 30 min had passed. Pool equilibration of the radioactive precursors was not limiting to these measurements. Polyadenylated RNA was isolated from dormant spores by oligodeoxythymidylic acid-cellulose chromatography and was found to comprise 3.3% of the total cellular RNA. Sucrose density gradient centrifugation revealed the polyadenylated RNA to be heterodisperse in size, ranging from 6S to 20S. It was concluded that M. racemosus sporangiospores contain preformed mRNA which is translated commencing immediately upon the addition of nutrient medium.

Fungal Proteins↗
PubMed1981

Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries.

A metalloendopeptidase optimally active at a neutral pH was purified 10000-fold from particulate fractions of bovine pituitaries. The solubilized enzyme has an apparent molecular weight of about 90 000, as determined by gel filtration on Sephadex G-200 and G-100 columns. The enzyme is not sensitive to inhibition by SH-blocking agents, diisopropyl fluorophosphate, leupeptin, pepstatin, antipain, and chymostatin. Thiols and metal chelators such as ethylenediaminetetraacetic acid (EDTA) o-phenanthroline are inhibitory. An EDTA-treated enzyme can be reactivated by several divalent metal ions, with zinc giving reactivation at the lowest concentrations. The specificity and kinetic parameters of the enzyme were studied with a series of synthetic peptide naphthylamides. The enzyme cleaves bonds in which the amino group is provided by a hydrophobic amino acid residue (position P1'). Replacement of this residue by small neutral amino acids decreases or virtually eliminates activity. The nature of substituents in positions P1, P2, P3, and P4 greatly influences specificity. Relatively high kcat and kcat/Km ratios were obtained with substrates containing arginine residues in positions P1 and P2. In such cases the impression of a "trypsin-like" activity was created. High reaction rates were also observed with substrates containing small neutral amino acids in positions P1 and P2, provided that position P3 was occupied by the acidic (polar) glutaryl residue. Replacement of this residue with hydrophobic substituents greatly decreased the rate of reaction. When positions P1 and P2, however, were occupied by arginine residues, the unfavorable effect of hydrophobic substituents in position P3 or P4 on catalysis was eliminated.

Animals↗
PubMed1981

Growth-rate-dependent adjustment of ribosome function in the fungus Mucor racemosus.

The dimorphic fungus Mucor racemosus was grown at rates between 0.043 and 0.434 doubling/h while maintained as yeasts or at rates between 0.21 and 0.50 doubling/h while maintained as hyphae by altering the composition of the growth medium or the gaseous environment of the cells. Yeasts at the higher growth rates contained many more ribosomes than did yeasts at the lower growth rates. They also had a higher percentage of ribosomes active in protein synthesis and a faster rate of polypeptide-chain elongation than did the slower-growing cells. Hyphal cells at faster growth rates also contained many more ribosomes and showed a faster rate of polypeptide-chain elongation than did slower-growing cells. However, the faster-growing cells had a substantially lower proportion of ribosomes active in protein synthesis than did the slower-growing hyphae. Pulse-chase experiments failed to provide any evidence of protein turnover, which might otherwise invalidate the values calculated for the peptide-chain elongation rates.

Kinetics↗
PubMed1981

Relationship of internal cyclic AMP levels, rates of protein synthesis and mucor dimorphism.

Changes in the intracellular pools of cyclic AMP and specific rates of protein synthesis have been described as correlates of the yeast-to-hypha conversion in Mucor racemosis. A further examination of the relationship between these physiological parameters and the cellular morphogenesis was conducted in the present study. The levels of intracellular cyclic AMP consistently varied as a function of the cell morphology rather than the CO2 tension, oxygen tension or growth rate. The specific rate of protein synthesis failed to change during a N2-to-air-induced yeast-to-hypha transition. Previously reported changes in this parameter during CO2-to-air- and CO2-to-N2-induced yeast-to-hypha shifts may be a consequence of growth rate changes rather than development per se.

Carbon Dioxide↗
PubMed1981

Proteolytic enzymes in bronchopulmonary lavage fluids: cathepsin B-like activity and prolyl endopeptidase.

Two proteolytic enzymes, a cathepsin B-like activity and prolyl endopeptidase activity, were demonstrated in cellular elements and fluids obtained from human bronchopulmonary washings and in fragments of human lung tissue. Model synthetic substrates were used for the determination of enzyme activities. Cathepsin B-like activity was determined with N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-arginyl-2-naphthylamide. The rate of reaction with this substrate was 300 times greater than the rate with alpha-N-benzoyl-DL-arginyl-2-naphthylamide, a substrate commonly used for the determination of cathepsin B activity. Prolyl endopeptidase was determined with N-benzyloxycarbonyl-glycyl-L-prolyl-sulfamethoxazole as the substrate. The two model substrates make possible the determination of enzyme activities in as little as 10 to 50 microliters of lavage fluid. High cathepsin B-like activity and prolyl endopeptidase were generally associated with high macrophage counts in lavage fluids. This observation and the findings that cathepsin B activity is more than 4.5 times higher in normal monocytes than in neutrophils and that prolyl endopeptidase activity could not be detected in normal neutrophils suggest that the enzymes in lavage fluids are mainly derived from macrophages. Large differences in cathepsin B-like activity in certain lavage fluids containing similar macrophage counts suggest the possibility of enzyme induction in these cells. High cathepsin B-like activity and prolyl endopeptidase were also found in lavage fluids after removal of cells by centrifugation. The possibility that these enzymes affect collagen turnover and lung remodeling needs to be explored.

Adult↗